1. Field of the Invention
The present invention relates to compositions for the protection, treatment, repair and reduction of inflammation of connective tissue in humans and animals and, in particular, to compositions capable of promoting anti-inflammation, chondroprotection, chondromodulation, chondrostabilization, chondrometabolization and the repair and replacement of human and animal connective tissue.
2. Background of the Invention
The connective tissues of humans and animals are constantly subjected to stresses and strains from mechanical forces and from diseases that can result in afflictions, such as arthritis, joint inflammation and stiffness. Indeed, connective tissue afflictions are quite common, presently affecting millions of Americans. Further, such afflictions can be not only painful but, in their extreme, debilitating.
The treatment of connective tissue afflictions can be quite problematic. A simple decrease in the stress to which the connective tissue is subjected is often not an option, especially in the case of athletes and animals such as race horses. Consequently, treatment is often directed at controlling the symptoms of the afflictions and not their causes, regardless of the stage of the degenerative process.
Presently, steroids, such as corticosteroids and NSAIDs, are widely used for the treatment of these ailments. [Vidal, et al., Pharmocol. Res. Commun., 10:557-569 (1978)]. However, drugs such as these, which inhibit the body's own natural healing processes, may lead to further deterioration of the connective tissue.
Connective tissue, for example articular cartilage, is naturally equipped to repair itself by manufacturing and remodeling prodigious amounts of collagen (a chief component of connective tissue such as cartilage) and proteoglycans (PGs) (the other major component of connective tissue such as cartilage). This ongoing process is placed under stress when an injury occurs. In such cases, the production of connective tissue matrix (collagen and PGs) can double or triple over normal levels, thereby increasing the demand for the building blocks of both collagens and proteoglycans.
The building blocks for collagen are amino acids, especially proline, glycine and lysine. PGs are large and complex macromolecules comprised mainly of long chains of modified sugars called glycosaminoglycans (GAGs) or mucopolysaccharides. The terms GAGs and mucopolysaccharides are understood in the art to be interchangeable. PGs provide the framework for collagen formation and also hold water to give flexibility, resiliency and resistance to compression.
Like almost every biosynthetic pathway in the body, the pathways by which both collagen and GAG form single molecule precursors are quite long. As is also characteristic of other biosynthetic pathways, the pathways by which collagen and GAGs are produced include what is called a rate-limiting step--that is, one highly regulated control point beyond which there is a commitment to finish. The presence of such rate-limiting steps permits complicated biosynthetic processes to be more easily and efficiently controlled by permitting the organism to focus on one point. For example, if conditions demand production and all the requisite raw materials are in place, then stimulation of the rate-limiting step will cause the end product to be produced. To stop or slow production, the organism needs simply to regulate the rate-limiting step.
In the production of PGs, the rate-limiting step is the conversion of glucose to glucosamine for the production of GAGs. Glucosamine, an aminosugar, is the key precursor to all the various modified sugars found in GAGs, including glucosamine sulfate, galactosamine, N-acetylglucosamine, etc. Glucosamine also makes up to 50% of hyaluronic acid--the backbone of PGs--on which other GAGs, like chondroitin sulfate are added. The GAGs are then used to build PGs and, eventually, connective tissue. Once glucosamine is formed, there is no turning away from the synthesis of GAG polymers.
Glucosamine has been shown to be rapidly absorbed into humans and animals after oral administration. A significant portion of the ingested glucosamine localizes to cartilage and joint tissues, where it remains for long periods. This indicates that oral administration of glucosamine reaches connective tissues, where glucosamine is incorporated into newly-synthesized connective tissue.
Glycosaminoglycans and collagen are the chief structural elements of all connective tissues. Their synthesis is essential for proper maintenance and repair of connective tissues. In vitro, the introduction of glucosamine has been demonstrated to increase the synthesis of collagen and glycosaminoglycans in fibroblasts, which is the first step in repair of connective tissues. In vivo, topical application of glucosamine has enhanced wound healing. Glucosamine has also exhibited reproducible improvement in symptoms and cartilage integrity in humans with osteoarthritis. [L. Bucci, Nutritional Supplement Advisor, (July 1992)].
The pathway for the production of proteoglycans may be briefly described as follows. Glucosamine is the main building block of connective tissue and may be provided either through the enzymatic conversion of glucose or through diet or external administration (see FIG. 1). Glucosamine may be converted into the other main component of connective tissue, namely PGs, upon incorporation of glucosamine into GAGs (see FIG. 2).
More specifically, GAGs are large complexes of polysaccharide chains associated with a small amount of protein. These compounds have the ability to bind large amounts of water, thereby producing a gel-like matrix that forms the body's ground substance. GAGs stabilize and support cellular and fibrous components of tissue while maintaining the water and salt balance of the body. The combination of insoluble protein and the ground substance forms connective tissue. For example, cartilage is rich in ground substance while tendon is composed primarily of fibers.
GAGs are long chains composed of repeating disaccharide units of monosaccharides (aminosugar-acidic sugar repeating units). The aminosugar is typically glucosamine or galactosamine. The aminosugar may also be sulfated. The acidic sugar may be D-glucuronic acid or L-iduronic acid. GAGs, with the exception of hyaluronic acid, are covalently bound to a protein, forming proteoglycan monomers. These PGs consist of a core protein to which linear carbohydrate chains formed of monosaccharides are attached. In cartilage proteoglycan, the species of GAGs include chondroitin sulfate and keratin sulfate. The proteoglycan monomers then associate with a molecule of hyaluronic acid to form PG aggregates. The association of the core protein to hyaluronic acid is stabilized by link proteins.
The polysaccharide chains are elongated by the sequential addition of acidic sugars and aminosugars, and the addition is catalyzed by a family of transferases. Aminosugars, such as glucosamine, are synthesized through a series of enzymatic reactions that convert glucose to glucosamine, or alternatively may be provided through the diet. The glucosamine is then incorporated into the GAGs as described above. Acidic sugars may be provided through the diet, may be obtained through degradation of GAGs by degradative enzymes, or produced through the uronic acid pathway.
Since repeating disaccharide units contain one aminosugar (such as glucosamine), it is clear that the presence of an aminosugar in the production of connective tissue is important. Glucosamine is, by far, the more important ingredient in the production of connective tissue since it is the essential building block of GAGs. See FIG. 1. GAGs contain hexosamine or uronic acid derivative products of the glucose pathway and from exogenous glucosamine, for example:
Hyaluronic acid Glucosamine + Glucuronic Acid Keratan Sulfate Glucosamine + Galactose Chondroitin Sulfate Glucuronic Acid + Galactosamine Heparin Sulfate Glucosamine + Glucuronic or Iduronic Acid Heparan Sulfate Glucosamine + Glucuronic or Iduronic Acid Dermatin Sulfate Iduronic Acid + Galactosamine
Chondroitin sulfate is a GAG that provides a further substrate for the synthesis of the proteoglycans. The provision of the chondroitin in its salt (e.g., sulfate) form facilitates its delivery and uptake by the humans and animals in the production of connective tissue. In addition, the sulfate portion of chondroitin sulfate is available for use in catalyzing the conversion of glucosamine to GAGs. Fragments of GAGs, including chondroitin sulfate, may also be used to provide a substrate for synthesis of proteoglycans since the assembly of PG occurs in the extracellular space.
In addition, chondroitin sulfate has been shown to have cardiovascular health benefits. [Morrison et al., Coronary Heart Disease and the Mucopolysaccharides (Glycosaminoglycans), pp. 109-127 (1973)]. Thus, the preferred form of glycosaminoglycan included in the compositions of the present invention is chondroitin sulfate or fragments thereof.
Chondroitin may be more efficacious than glucosamine for injury rehabilitation. [Christensen, Chiropractic Products, pp. 100-102 (April 1993)]. An evaluation of glucosamine versus chondroitin for treatment of osteoarthritis has been conducted and concludes, contrary to Christensen, that glucosamine is preferred. [Murray, MPI's Dynamic Chiropractic, pp. 8-10 (Sep. 12, 1993)]. Neither reference teaches or suggests combining the materials. Bucci (Townsend Letter for Doctors, pp. 52-54, January 1994), discloses the combination of glucosamine and chondroitin for treatment of osteoarthritis. Bucci has acknowledged that this combination was personally disclosed to him by one of the present inventors.
Chondroitin sulfate also acts to inhibit the degradative enzymes that break down connective tissue. In so doing, chondroitin sulfate promotes the maintenance of healthy connective tissues. When combined with glucosamine, which functions primarily as a building block for the synthesis of connective tissue, chondroitin sulfate works in concert with the glucosamine but may work in a different fashion. The ability of chondroitin sulfate to block degradation is one of its important functions.
GAGs may be semi-synthetic in that they may be chemically modified to contain more sulfur groups than in their initially extracted form. In addition, GAGs may be partially or completely synthesized, and therefore its building blocks (and, hence, the compound itself) may be of either plant or animal origin.
Pentosan polysulfate (PPS) is a compound that is understood to have anti-inflammatory activity, increase chondrocyte macromolecule biosynthesis resulting in increased cartilage matrix replacement, stimulate synovial fibroblast biosynthesis of hyaluronic acid, inhibit enzymes implicated in the degradation of cartilage matrix, mobilize thrombi and fibrin deposits in synovial tissues and subchondral blood vessels thereby increasing perfusion of the tissue, and mobilize lipids and cholesterol in synovial and subchondral blood vessels. As such, PPS is believed to accomplish many of the same functions as GAGs.
PPS is a semi-synthetic polysulfated xylan that is a sulfated form of a compound extracted from beechwood hemicellulose consisting of repeating units of (1-4) linked .beta.-D-xylano-pyranoses. PPS is a low molecular weight linear polysaccharide, a semi-synthetic heparinoid that is considered an oversulfated form of a GAG.
There are several forms of PPS that display the above-described activities. Sodium PPS and a calcium-derived PPS (called CAPPS) may both be used to accomplish the functions of PPS. Each of these forms of PPS exhibit GAG-like activity, and will hereinafter be referred to as GAG-like compounds.
S-Adenosylmethionine (SAM) is a significant physiologic compound which is present throughout body tissue and takes part in a number of biologic reactions as a methyl group donor or as an enzymatic activator during the synthesis and metabolism of hormones, neurotransmitters, nucleic acids, phospholipids, and proteins. SAM may be second only to adenosine triphosphate (ATP) in the variety of reactions in which it is a cofactor. SAM is metabolized via three metabolic pathways of transmethylation, transsulfuration, and aminopropylation. [Stramentinoli, Am. J. Med., 83(5A):35-42 (1987)]. In higher organisms, SAM plays a significant role in transmethylation processes with more than 40 anabolic or catabolic reactions involving the transfer of the methyl group of SAM to substrates such as nucleic acids, proteins, and lipids, among others. Also, the release of the methyl group from SAM is the start of a "transsulfuration" pathway that produces all endogenous sulfur compounds. After donating its methyl group, SAM is converted into S-adenosylhomocysteine, which in turn is hydrolyzed to adenosine and homocysteine. The amino acid cysteine may then be produced from the homocysteine. The cysteine thus produced may exert a reducing effect by itself or as an active part of glutathione, which is a main cell anti-oxidant. [Stramentinoli, cited above].
SAM has been used to treat various disorders. In various forms of liver disease, SAM acts as an anticholestatic agent. [Adachi et al., Japan Arch. Inter. Med., 33:185-192 (1986)]. SAM has also been administered as an antidepressant for use in the management of psychiatric disorders [Caruso et al., Lancet, 1: 904 (1984)], and as an anti-inflammatory compound in the management of osteoarthritis [Domljan et al., Int. J. Clin. Pharm. Toxicol., 27(7):329-333 (1989)].
Low levels of SAM are believed to play a role in reducing the risk of certain cancers. [Feo et al., Carcinogenesis, 6:1713-20 (1985)]. In addition, the administration of SAM has also been associated with a fall in the amount of early reversible nodules and the prevention of the development of late pre-neoplastic lesions and hepatocellular carcinomas. [Garcea et al., Carcinogenesis, 8:653-58 (1987)].
Unfortunately, SAM per se is unstable due to its high reactivity. The relatively recent synthesis of stable salts, however, has made SAM available for research and therapeutic use. [See, e.g., U.S. Pat. Nos. 4,990,606 and 5,102,791].
SAM has been used outside of the United States in a number of clinical trials concerning the treatment of osteoarthritis. While used in these trials primarily as an analgesic and replacement for NSAID therapy, SAM is a precursor of polyamines. In addition to their analgesic and anti-inflammatory properties, and their ability to scavenge free radicals, polyamines may stabilize the polyanionic macromolecules of proteoglycans. [Schumacher, Am. J. Med., 83(5A):2 (1987)].
SAM may also function as a source of endogenous sulfur, which will increase sulfation of GAGs to be incorporated in proteoglycans. The inclusion of SAM is particularly beneficial in instances of subclinical deficiencies of SAM, occurring especially in elderly populations with higher risk of osteoarthritis [Frezza et al., Gastroenterol., 99:211-215 (1990)]. The supplementation of SAM may aid in instances of SAM deficiency where the ability of the body to sulfate GAGs may be compromised.
In addition, a number of metabolites of SAM aid in the repair of connective tissue, including glutathione, polyamines, methylthioadenosine, and adenosine. Glutathione works as a scavenger of oxygen-related products [Shumacher, Am. J. Med., 83(Supp 5a):1-4 (1987); Matthew & Lewis, Pharmacol. (Life Sci. Adv.), 9:145-152 (1990); Szabo et al., Science, 214:200-202 (1981)] and thus has an anti-inflammatory effect. Polyamines, including spermine, spermidine, and putrescine, stabilize polyanionic macromolecules of proteoglycans [Schumacher, cited above; Conroy et al., Biochem. J., 162:347-350 (1977)] and thus protect proteolytic and glycolytic enzymes. These polyamines also have an anti-inflammatory effect [Bird et al., Agents Actions, 13:342-347 (1983); Oyangui, Agents Actions, 14:228-237 (1984)], probably as a scavenger of oxygen-related products [Kafy et al., Agents Actions, 18:555-559 (1986); Matthews & Lewis, cited above], and have an analgesic effect [Bird et al., cited above; Oyangui, cited above]. The SAM metabolite methylthioadenosine has a pronounced anti-inflammatory effect [Matthews & Lewis, 1990] while adenosine has a more modest anti-inflammatory effect [Matthews & Lewis, 1990].
Studies have shown that some forms of exogenous SAM are stable in digestive juices when given orally. [Stramentinoli et al., cited above; Vendemiale et al., Scand. J. Gastroenterol., 24:407-415 (1989)]. The metabolism of exogenous SAM appears to follow known pathways of endogenous SAM metabolism. [Kaye et al., Drugs, 40(Suppl. 3):124-138 (1990)]. In humans, oral SAM was tolerated to the same extent as placebo with very mild nonspecific side effects. [Schumacher, cited above; Frezza et al., cited above].
Manganese plays a role in the synthesis of GAGs, collagen and glycoproteins which are important constituents of cartilage and bone. Manganese is important for enzyme activity of glycosyltransferases. This family of enzymes is responsible for linking sugars together into glycosaminoglycans, adding sugars to other glycoproteins, adding sulfate to aminosugars, converting sugars into other modified sugars, and adding sugars to lipids. The enzymatic functions of glycosyltransferases are important in glycosaminoglycan synthesis (hyaluronic acid, chondroitin sulfate, keratan sulfate, heparin sulfate and dermatin sulfate, etc.), collagen synthesis, and in the functions of many other glycoproteins and glycolipids.
Manganese also plays a role in the synthesis of glycosaminoglycans and glycoproteins, which are important constituents of cartilage and bone. Many reproductive problems in horses and skeletal abnormalities in foals have been ascribed to manganese deficiency. [Current Therapy in Equine Medicine, 2:402-403 (1987)].
Manganese deficiency leads to abnormal bone growth, swollen and enlarged joints, and slipped tendons in humans and animals. In humans, manganese deficiencies are also associated with bone loss and arthritis. Levels of all glycosaminoglycans are decreased in connective tissues during manganese deficiencies, with chondroitin sulfates being most depleted. Manganese-deficient organisms quickly normalize glycosaminoglycans and collagen synthesis when manganese is replenished.
Approximately 40% of dietary manganese is absorbed by the body tissue. Storage of manganese in the body is minimal--a mere 12 to 20 mg is present in the body at any one time. Large amounts of calcium and phosphorus in the intestine are also known to interfere with manganese absorption. The richest dietary sources are the foods least consumed by the general public, such as whole grain cereals and breads, dried peas, beans and nuts. The ascorbate form of manganese is preferred due to the high bioavailability and the need for vitamin C (ascorbic acid) for collagen production. Vitamin C also enhances manganese uptake by the body.
Other optional ingredients in the compositions of the present invention are methyl donors or methyl donor cofactors, such as vitamins B.sub.12 and B.sub.6, folic acid, dimethylglycine, trimethylglycine, and betaine. These ingredients augment the function of SAM in that they are cofactors in methylation or stimulate the production of endogenous SAM. [See, e.g., A. Barak et al., Alcoholism: Clin. and Exp. Res., 17(3):552-555 (1993)]. In addition, these compounds are likely to be lacking in patients suffering from connective tissue disorders. For example, it is estimated that 12% of the elderly population in the United States suffers from a vitamin B.sub.12 deficiency, a group more likely to suffer from connective tissue disorders.
An adequate amount of vitamin B.sub.12, for example, has an important environmental influence on the accumulation of homocysteine that results from the metabolism of SAM. In other words, methyl donors or methyl donor cofactors, such as vitamin B.sub.12 and the others listed in the preceding paragraph, can reduce levels of homocysteine when administered either alone or in combination.
Vitamin B.sub.12 is generally known to function as a coenzyme in biochemical reactions such as the synthesis of proprionic acid and of methionine. Recent evidence suggests that the elevated levels of plasma homocysteine increase the risk of occlusive vascular disease. Adequate amounts of vitamin B.sub.12 are considered the most important environmental influence on the accumulation of unnecessary homocysteine. [Joosten et al., Am. J. Clin. Nutr., 58(4): 468-76 (1993)]. In addition, it is also understood that vitamin B.sub.12 may play a role in the methylation of selenium. [Chen and Whanger, Tox. and Appl. Pharm., 118:65-72 (1993)]. Specifically, increased levels of vitamin B.sub.12 significantly contribute to selenium methylation and might decrease overall selenium toxicity by preventing its accumulation in tissues. [Chen and Whanger, cited above].